Psychosis protecting nucleic acid, peptides, compositions and method of use

ABSTRACT

Psychosis protecting (PP) nucleic acids and encoded PP peptides and related proteins, and antibodies, anti-idiotype antibodies, and fragments thereto, for treatment, diagnosis and/or research related to the protection from psychosis such as schizophrenia or related disorders, or symptoms thereof, and expression products, compositions and methods therefor, including treatment of schizophrenia and related disorders, as well as transgenic non-human mammals epxressing PP peptide or related protein encoding nucleic acids.

This invention was made with Government support under MH 35976 and MH08618 awarded by the National Institute of Mental Health. The Governmentthus has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a 35 USC 371 application of PCT/US94/05445,filed May 13, 1994, and a continuation-in-part of application No.08/060,560, filed May 13, 1993, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of molecular biology andmedicine, and more particularly to psychosis protecting (PP) nucleicacids and peptides involved in protection from psychoses and relateddisorders, as well as expression products, compositions thereof, andmethods therefor, including detection, amplification, isolation andexpression of such PP nucleic acids and PP peptides, as well asdiagnostic and therapeutic methods using such PP peptides and theirencoding PP nucleic acid.

2. Background of the Related Art

Psychoses, such as schizophrenia can be differentiated into two basiccategories; those which are amenable to treatment, by means ofconventional antipsychotic drugs, and those which are resistant totreatment, the latter usually being spoken of as "chronic" or "negativesymptom" schizophrenia. Preclinical conditions of psychoses are alsoprevalent and could be subject to treatment if the degree of severitycould be diagnosed in a standardized manner. These categories can, tosome degree, be correlated with the relative balance of positive andnegative symptomatology. The designation "negative (Bleulerian)symptomatology", although long known, has in recent years been used moreroutinely.

Treatment of psychoses and schizophrenia. Treatment of schizophrenia andother psychoses is commonly provided using the antipsychotics termedneuroleptic agents. Neuroleptic agents, regardless of their chemicalstructures, are pharmacologically active upon the dopamine receptorsystem, as dopamine antagonists. Many of these compounds, particularlythe phenothiazines, also have significant activity on otherneurotransmitter systems, in particular various serotonin subtypes,particularly the 5-HT-2, and on muscarinic receptors,alpha-adrenoceptors, or histamine H-1 or H-2 receptors. The clinical useof neuroleptics has provided a means for treating patients sufferingfrom psychotic disorders, including schizophrenia. Short-term use ofneuroleptics is indicated in several types of exacerbations ofschizophrenia. Continuous long-term use of neuroleptics is indicated,e.g., in primary indications involving schizophrenia as well asquestionable indications such as chronic characterological disorderswith schizoid, "borderline," or neurotic characteristics. See, e.g.,Baldessarini, Chemotherapy in Psychiatry, Revised and Enlarged Edition,Harvard University Press, Cambridge, Mass., (1985), the contents ofwhich are entirely incorporated herein by reference.

Neuroleptics and Their Side Effects. Neuroleptics are also referred toas neuroplegics, psychoplegics, psycholeptics, antipsychotics and majortranquilizers, but are sometimes distinguished from non-neurolepticpsychotropics. Neuroleptics have also been characterized as agents thatproduce sedative or tranquilizing effects, and which also produce motorside effects, such as catalepsy or extrapyramidal symptomatology.Nonlimiting representative examples of neuroleptics includephenothiazine derivatives (e.g., chlorpromazine); thioxanthinederivatives (e.g., thiothixene); butyrophenone derivatives (e.g.,haloperidol); dihydroindolone (e.g., molindone); dibenzoxazepinederivatives (e.g., loxapine); and "atypical" neuroleptics (e.g.,sulpiride, remoxipride pimozide and clozapine). See Berstein ClinicalPharmacology Littleton, Mass.:PSG Publishing (1978); Usdin et alClinical Pharmacology in Psychiatry New York:Elsevier North-Holland(1981); and Baldessarini, supra, (1985); which references are hereinentirely incorporated by reference.

The long term use of all known anti-psychotics, including neuroleptics,has resulted in serious side effects, as set forth in Table I, such aspersistent and poorly reversible motoric dysfunctions (e.g., tardivedyskinesia) in a significant number of patients. For example, classicalneuroleptic agents, as exemplified by the butyrophenones andphenothiazines, can, upon long-term administration, produce severemotoric symptomatology, termed tardive dyskinesia. These motor movementsare uncontrollable and can range from relatively trivial manifestationsto total debilitation. Tardive dyskinesia is usually reversible upondiscontinuation of the chronic neuroleptic, if the drug is discontinuedsoon after symptoms of tardive dyskinesia appear. Otherwise symptoms maypersist. Pharmacological intervention for treatment of tardivedyskinesia is only moderately successful. Such motor abnormalities areknown to occur in as high as 10% of the patients who are maintained onthese drugs for several years; the incidence is much greater in certaingroups, such as middle-aged females.

The following Table I presents these and additional neurological sideeffects of neuroleptic anti-psychotic drugs.

                                      TABLE I    __________________________________________________________________________    Neurological Side Effects of    Neuroleptic-Antipsychotic Drugs                      Period of    Reaction           Features   maximum risk                             Proposed mechanism                                       Treatment    __________________________________________________________________________    Acute dystonia           Spasm of muscles of                      1-5 days                             Dopamine excess?                                       Antiparkinsonism agents are           tongue, face, neck,                             Acetylcholine excess?                                       diagnostic and curative           back; may mimic             (i.m. or i.v., then p.o.)           seizures; not           hysterical    Parkinsonism           Bradykinesia, rigidity,                      5-30 days                             Dopamine blockade                                       Antiparkinsonism agents           variable tremor, mask-                      (rarely          (p.o); dopamine agonists           facies, shuffling gait                      persists)        risky?    Akathisia           Motor restlessness;                      5-60 days                             Unknown   Reduce dose or change drug           patient may experience                      (commonly        low doses of propranolol;.sup.a           anxiety or agitation                      persists)        antiparkinsonism agents or                                       or benzodiazepines may help    Tardive           Oral-facial dyskinesia;                      6-24 months                             Dopamine excess?                                       Prevention best; treatment    dykinesia           choreo-athetosis; some-                      (worse on        unsatisfactory; slow    spontaneous           times irreversible,                      withdrawal)      remission           rarely progressive    "Rabbit"           Perioral tremor (late                      Months or                             Unknown   Antiparkinsonism agents; reduce    syndrome           parkinsonism variant?);                      years            dose of neuroleptic           usually reversible    Malignant           Catatonia, stupor,                      Weeks  Unknown   Stop neuroleptic; antiparkinsonism    syndrome           fever, unstable pulse       agents usually fail; bromocriptine           and blood pressure;         often helps; denatrolene variable;           myoglobinema; can           general supportive care crucial           be fatal    __________________________________________________________________________

In addition, clozapine, although apparently capable of producing lessmotor side effects, can cause irreversible, potentially fatalagranulocytosis in a minority of patients administered the drug. Suchserious side effects limit the use of clozapine to patients who areresistant to treatment with other neuroleptics.

These side effects are especially prevalent in geriatric populations,and adequate pharmacological treatment of these debilitating motoricdysfunctions is not currently available. This problem has been generallyassociated with long-term, clinical administration of these agents,including their use in the long term treatment of schizophrenia. Thereis thus a great need for alternative treatments for schizophrenia,including chronic schizophrenia, without toxic side effects of knownagents used for such treatment, or whose long-term administration willnot produce such toxic side effects.

Treatments proposed for schizophrenia. Anti-psychotic drugs, such asneuroleptics have been found to generally affect neuroreceptors, such asdopamine and serotonin receptors. Many of these receptors have beenrecently cloned and sequenced, such as the serotonin 5-HT1 and 5-HT2(see, e.g., Leonard, Int. Clin. Psychopharmacol., 7(1):13-21 (1992)) anddopamine receptors: D5 (Sunahara et al., Nature, 350:614-619 (1991)); D4(Van Tol et al., Nature, 350:610-614 (1991)); D1 (Zhou et al., Nature,347:76-80 (1990); Dearry et al., Nature, 347:72-76 (1990)); and rat D2(Tourtellotte et al., Neurochem. Res., 12:565-571 (1987); Bunzow et al,Nature, 33:783-787; Miller et al, Biochem. Biophys. Res. Com.,166:109-112)).

Currently anti-psychotic agents (neuroleptics) are used for thetreatment of schizophrenia and all other psychoses. Proposed treatmentsinvolve the use of compositions containing peptides and proteins whichmay act as ligands for receptors or portions of receptors as well asother neural active peptides and analogs thereof. Examples of suchcompositions include neurotensin peptide analogs (WO 93/00359, Du PontMerck Pharmaceutical Co. (1993)), tachykinin agonists (WO 92/22569,Fujisawa Pharmaceutical Co., LTD. (1992); EP 482 539, FujisawaPharmaceutical Co., LTD. (1992)), galanin agonists (WO 92/20709, AstraAB (1992)), neurokinin receptor and fragments (WO 92/16547, Children'sMedical Center (1992); dopamine receptor agonist/antagonist peptides (WO91/04271, BASF AG, (1991)), thyrotropin releasing hormone analogs (U.S.Pat. No. 5,098,888, Vincent et al (1992)), enkephalin like peptides (WO90/00564, Research Corp. Techn., Inc. (1990); U.S. Pat. Nos. 4,684,620(1987) and 4,518,711 (1985), Hruby et al; EP 050 828, Merck, Inc.(1984)), calmodulin binding peptides (U.S. Pat. No. 5,182,262, Hruby(1993)), cerulein peptides (U.S. Pat. No. 4,552,865, Fujino et al(1985)), and dopamine releasing protein (U.S Pat. No. 5,149,786, Marcuset al. (1992)).

Citation of documents herein is not intended as an admission that any ofthe documents cited herein is pertinent prior art, or an admission thatthe cited documents are considered material to the patentability of anyof the claims of the present application. All statements as to the dateor representation as to the contents of these documents is based on theinformation available to the applicant and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome one or moredeficiencies of the related art.

It is another object of the present invention to provide a psychosisprotecting gene and expression products thereof, such as psychosisprotecting (PP) peptides or psychosis protecting nucleic acids that areexpressed in normal people, but not expressed in patients diagnosed withclinical schizophrenia or other psychoses.

It is a further object of the present invention to provide methods fordetecting the relative lack of expression of a psychosis protecting genein a mammal, having a maximum range of expression that correlates withat least one clinical symptom associated with a psychotic disorder.These symptoms of various psychoses include thought disorders, affectualblunting, delusions, hallucinations, anhedonic cognitive impairment.

It is yet a further object of the present invention to provide a meansfor diagnosing impending psychosis in individuals at risk forschizophrenia or other psychoses who do not express genes or DNAcomprising a sequence corresponding to a PP peptide or related peptideor protein.

It is yet another object of the present invention to use a portion of anucleic acid sequence corresponding to a DNA sequence according to SEQID NO:3 as a probe to obtain and/or sequence a full length gene asexpressed in lymphocytes or other accessible tissues and in brain tissuefrom psychotic and normal individuals or animals.

Another object of the present invention is to use the gene fragment asdescribed in SEQ ID NO:3 to identify the DNA that corresponds to a PPpeptide or protein and to identify the full length DNA that representsan extension of the present gene fragment.

It is also an object of the present invention to make an animal model ofpsychosis or animals vulnerable to psychosis by inhibiting theexpression of PP peptide related proteins in rats, mice or othernon-human species. In one aspect, a transgenic experimental animal isprovided which has been transformed by the gene carrying the nucleicacid sequences inhibiting the expression of PP peptide related proteinsso as to obtain an animal model exhibiting psychotic symptoms and thecorresponding neurophysiology. One example of such an inhibiting nucleicacid sequence encoding an anti-sense nucleic acid which is complementaryto the DNA sequence of (SEQ ID NO:3).

The present invention is also directed to a transgenic laboratory animalas a model of a psychotic disorder which is produced by inserting a PPpeptide related protein inhibiting nucleic acid of this invention into amouse or other suitable laboratory animal so that the animal displayspsychotic symptoms corresponding to a known psychotic disorder. Such ananimal model enables testing on non-humans of treatment and diagnosticmethods for psychotic disorders, such as schizophrenia, schizoaffectivedisorders, paranoid disorders, and some mood disorders.

It is also an object of the present invention to enable geneticcounselors to provide information about the risk of schizophrenia orother psychoses by determining whether the protective gene describedherein is actively expressed in an individual at risk for psychosis.

It is also an object to provide methods for treating psychoses byproviding expression or expression products of a psychosis protectinggene as therapeutic compounds, compositions and methods.

It is another object of the present invention to provide monoclonalantibodies, anti-idiotype antibodies, or fragments thereof, whichspecifically bind an epitope of a psychosis protecting peptide.

It is yet another object of the present invention to provide PPpeptides, antibodies, anti-idiotype antibodies, compositions and methodsthat can be used in therapeutic and/or diagnostic applications forpsychosis, due to their expected biological properties.

A further object of the present invention is to provide synthetic,isolated or recombinant peptides which are designed to inhibit or mimicvarious PPs or fragments thereof, which are effective for the treatmentor diagnosis of symptoms relating to schizophrenia or other psychoses.

It is another object of the present invention to provide non-naturallyoccurring synthetic, isolated and/or recombinant PP peptides which arefragments and/or muteins of polypeptides encoded by the gene, a fragmentof which is the DNA sequence of (SEQ ID NO:3), or at least one of SEQ IDNOS:4-12, which encoded PP peptides are expected to have therapeuticeffects in psychotic patients and which are useful for providingdiagnostic, therapeutic or research compounds, compositions and methodsof use.

In a preferred embodiment, the peptide is (a) chemically synthesizedand/or (b) obtained from a recombinant host cell or organism whichexpresses a recombinant nucleic acid encoding a PP peptide, as definedherein, and/or may be provided as a therapeutic or diagnostic nucleicacid.

In another aspect of the present invention, a PP composition isprovided, comprising at least one PP peptide, or a pharmaceuticallyacceptable ester, ether, sulfate, carbonate, malate, glucuronide or saltthereof, the PP composition optionally further comprising apharmaceutically acceptable carrier and/or diluent.

In still another aspect of the present invention, a method is providedfor treating a subject suffering from symptoms associated withschizophrenia or any other psychotic disorder.

In a preferred embodiment, the PP peptide corresponds to an activeportion of a protein encoded by the genes, fragments of which are thenucleic acid sequences of SEQ ID NO:3, or SEQ ID NOs:4-12, wherein themethod comprises administering an effective psychosis treatingmodulating amount of a PP peptide of the present invention. In anotherpreferred embodiment, the disease state is a psychiatric disorderrelated to schizophrenia or schizo-affective disorder, or any otherpsychotic disorder, see American Psychiatric Association, Revised Manualof Diagnostic and Statistical Criteria for Psychiatric Disorders(DSM-III-R), American Psychiatric Assoc. Press, Washington, D.C. (1989),hereinafter "Criteria for Psychiatric Disorders" which is entirelyincorporated herein by reference.

In another preferred embodiment, the PP composition is administered as apharmaceutical composition to provide a PP peptide in an amount rangingfrom about 0.01 μg to 100 mg/kg, and also preferably, about 10 μg to 10mg/kg. In another preferred embodiment, the administeration is by oral,intravenous, intramuscular, parenteral or topical administration,including mucosal administration to the nasal mucosa or the oral mucosa,by aerosol, nebulizer or drop administration as non-limiting examples.

Other objects of the invention will be apparent to skilled practitionersfrom the following detailed description and examples relating to thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting a vector pOKSC4c.

FIG. 2 is a subtraction cloning scheme used to detect subtracted clonesused to obtain a schizophrenia protection gene according to the presentinvention.

FIGS. 3A-B are pictorial representations of in situ autoradiographiesshowing (FIG. 3A) hybridization of a psychosis protecting gene of thepresent invention with cortex and medial geniculate nuclei, includingthe CA1 to CA3 of Ammon's horn (hippocampus), with the interrhinal,perirhinal and temporal cortexes having higher signals; and (FIG. 3B)control showing absence of hybridization.

DETAILED DESCRIPTION OF THE INVENTION

Based on the discovery that normal monozygotic twins express a proteinwhich is not expressed in corresponding schizophrenic monozygotic twins,and which psychotic preventing (PP) peptide related protein is onlyfound to be expressed in areas of the cerebral cortex known to includeneurotransmission involved in psychotic disorders, the present inventionrelates to PP peptides corresponding to functional domains of thenormally expressed PP related protein. Such PP peptides are thusexpected to be used to mimic naturally occurring, PP peptide relatedproteins, which are expected to have a protective and/or therapeuticeffect on individuals suffering from symptoms relating to psychoses,such as schizophrenia or schizo-affective disorders or other psychoses(see, e.g., "Criteria for Psychiatric Disorders", supra).

The basis of the present invention was discovered in studies ofschizophrenia involving monozygotic twins where one twin has symptoms ofthe disease and the other twin is normal, which thus provides a"control" for the other twin. Estimates of concordance rates forschizophrenia in monozygotic twins vary, but are in the fifty percentrange. Inasmuch as both twins presumably have identical genes (verifiedby DNA fingerprinting) and immunological markers, the clinicalmanifestations of the illness might be determined by other factors aswell. If schizophrenia is of multifactorial etiology (i.e., havingmultiple gene and environmental components), the application ofquantitative genetic analysis may be inappropriate in the elucidation ofthe molecular etiology of the illness. One alternative approach is tostudy gene expression in affected individuals and controls.

Thus the present invention involved the subtraction cloning of cDNA frommRNA of such monozygotic twins to determine if a psychosis protectinggene was expressed in normal twins and not in psychotic twins. Thediscovery of such a PP peptide encoding gene, and confirmation ofexpression in the cortex where neurotransmission effects associated withpsychotic disorders has been determined, has provided a means to cloneand express such PP related proteins and related or functionally similarPP peptides, as well as antibodies thereto, which are expected to beuseful in the treatment, diagnosis and/or research involving psychoticdisorders in humans and animals.

Accordingly, a "psychosis protecting peptide" or "PP peptide" of thepresent invention includes peptides having a "PP amino acid sequence"which can be obtained initially by using the sequence presented in SEQID NO:3, or SEQ ID NOs:4-12, as a basis for designing polynucleotideprobes to clone, sequence and express or synthesize PP related proteinsand peptides occurring in normal individuals, and to a substantiallylesser degree in individuals with psychotic disorders, such aspolypeptides encoded in part by at least one gene, a fragment of whichis at least one nucleic acid sequence of SEQ ID NOs:3-12.

PP peptide nucleic acid probe detection of PP peptide epitope containingpeptides or proteins. PP peptide nucleic acid probes may be used todetect RNA or DNA encoding PP peptide related or homologous proteins asa means to diagnose or prediagnose psychosis or related disorders, suchas schizophrenia. Such nucleic acid probes may thus be used toquantitatively or qualitatively detect an RNA or DNA encoding a proteinor peptide corresponding at least in part to a PP peptide in a sample orto detect the presence of such nucleic acids in biological fluids orcells which express such nucleic acid, in vitro, in situ, or in vivo,based on the teaching and guidance presented herein, without undueexperimentation. The lack of, or presence of low concentrations of,nucleic acid encoding PP peptide related peptides and/or proteins isexpected to correlate with psychoses and related disorders, such asschizophrenia.

Nucleic acid probe assays capable of detecting the presence of such anucleic acid molecule, or proteins encoded therefrom, in a sample arewell known in the prediction and diagnosis of disease. Nucleic aciddetection assays can be predicated on any characteristic of the nucleicacid molecule, such as its size, sequence, susceptibility to digestionby restriction endonucleases, etc. Such a labeled, detectable probe canbe used by known procedures for screening a genomic or cDNA library of acell having a nucleic acid encoding a PP peptide related protein orpeptide or as a basis for synthesizing PCR or other nucleicamplification probes for amplifying a cDNA generated from an isolatedRNA encoding a target nucleic acid or amino acid sequence, as describedherein.

A detectably labeled oligonucleotide probe of this sort can be afragment of an oligonucleotide that is complementary to a polynucleotideencoding a PP peptide or fragment thereof. Alternatively, a syntheticoligonucleotide can be used as a target probe which is preferably atleast about 10 nucleotide in length (such as 10, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, or 100-423 or more, or any combination or range therein, inincrements of 1 nucleotide), such that the target probe is specific forthe desired nucleic acid sequence to be detected, amplified orexpressed. Preferably the nucleotide probe corresponds to at least aportion of a nucleic acid sequence presented in SEQ ID NO:3.

Nucleic acids, or protein encoded thereby, to be detected by a method ofthe present invention, may be contained in samples isolated from anytissue sample of an animal subject or patient, such as blood, lymph,saliva, urine, CNS, amniotic fluid, skin, hair, feces, or any othertissue, and analyzed by hybridization to labeled probes. Such probespreferably hybridize to PP peptide-encoding nucleotide under highstringency conditions or medium stringency conditions, depending on thepresence or possible presence of other non-target nucleic acids whichalso bind the probes specific for the target nucleic acids. For probedesign, hybridization, and stringency conditions, see, e.g., Ausubelsupra, sections 6.3 and 6.4, and Sambrook et al, supra.

A wide variety of such labels have been used for labeling detectableprobes, which can be used for labeling nucleic acid containing probes,as follows: (1) Kourilsky et al. (U.S. Pat. No. 4,581,333), e.g.,describe the use of enzyme labels to increase sensitivity in a detectionassay; (2) radioisotopic labels are disclosed, e.g., by Falkow et al.(U.S. Pat. No. 4,358,535), and by Berninger (U.S. Pat. No. 4,446,237);(3) fluorescent labels of probes can be used (e.g., Albarella et al., EP144914); (4) chemical labels of probes may be used (e.g., Sheldon III etal., U.S. Pat. No. 4,582,789, Albarella et al., U.S. Pat. No.4,563,417); (5) modified bases in the probes may be used (e.g., Miyoshiet al., EP 119448); (6) a restriction enzyme sensitive label fordifferential restriction endonuclease digestion may be used (Saiki etal., Biotechnology 3:1008-1012, 1985), (7) an allele specific labelusing allele specific oligonucleotide probes may be used Saiki et al,Nature 324:163-166 (1986), Conner et al., Proc. Nat'l Acad. Sci. USA,80:278 (1983), Holbeck and Nepom, Immunogenetics 24:251-258 (1986),Nepom et al, U.S. Pat. Nos. 5,039,606 and 4,971,902, and Whiteley et al,U.S. Pat. No. 4,833,750; (8) a ligase mediated label for ligase mediatedgene detection (LMGD) using oligonucleotide ligation assays may also beused (Landegren, et al., Science 241: 1077-1080, 1988), and (9) afluorescence energy transfer label for use in fluorescence resonanceenergy transfer (FRET), as disclosed, e.g., by Wolfe et al., Proc. Nat.Acad. Sci. USA 85: 8790-94 (1988)as non-limiting examples. See also,e.g., Ausubel et al, eds., supra; Sambrook, supra; Harlow, supra; andColigan et al., supra, For related technologies and methods. See also,e.g., Ausubel, supra, at §§9.5.2 (selectable markers), §9.8 (RNAanalysis), §§10.6-8 (detection of proteins), §§11.1-1.2 (immunoassays)and §§11.3.16 (preparation and use of monoclonal, polyclonal andantipeptide antibodies for protein detection). The above references areall entirely incorporated herein by reference.

Accordingly, detection of a nucleic acid encoding a PP peptide relatedprotein or peptide can be provided according to the present invention,based on the teaching and guidance presented herein, without undueexperimentation.

PP peptides of the present invention can include fragments and/or muteinpeptides encoded by the genes, fragments of which are the nucleic acidsequences of SEQ ID NO:3; FIG. 1 (SEQ ID NO: 1) or at least one of SEQID NOs: 4-12, or amino acids encoded thereby, biological activity whichmodulates one or more symptoms associated with schizophrenia orschizo-affective disorders, such as delusions, hallucinations(particularly arbitrary), thought disorder and emotional blunting, whichactivity is measurable in vitro, in vivo or in situ, using known testingas screening assays. In the context of the present invention,"anti-psychotic biological activities" refers to having a detectable ormeasurable improved effect on at least one psychosis associated symptom,such as improved behavior, thought process, speech, thought content,improved perceptual abnormalities, affect, cognitive functions, and thelike, as determined by known psychiatric evaluation techniques. See,.,e.g., Merck Manual, supra, Chs. 133-136 and 140-143; and Criteria forPsychiatric Disorders, supra, which are entirely incorporated byreference herein.

Alternatively or additionally, screening may be carried out using thegene fragment as at least a 10 nucleotide sequence appearing in SEQ IDNO:3 or at least one of SEQ ID NOs:4-12) as a probe in Northern analysisor for dot blot or slot blot or other techniques for detecting specificRNA or DNA sequences, e.g., as substantially corresponding to at leastone of SEQ ID NOs:3-12. Other methods for detecting the PP could also beused such as immunocytochemistry. Tissue sources of RNA could belymphocytes or other accessible tissues, or any tissue capable ofexpressing a PP peptide or PP nucleic acid.

PP peptides of the present invention can be synthesized or recombinantlyproduced, or optionally purified, to provide commercially useful amountsof PP peptides for use in therapeutic, diagnostic or researchapplications, according to known method steps, see, e.g., Ausubel et al,eds. Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, N.Y., N.Y. (1987, 1993); Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988);Sambrook et al, Molecular Cloning, A Laboratory Manual, 2nd edition,Vols. 1-3, Cold Spring Harbor Press, (1989); Coligan et al., eds.,Current Protocols in Immunology, Greene Publishing Associates and WileyInterscience, New York, N.Y., (1992, 1993), which references are hereinentirely incorporated by reference.

Additionally, PP peptides according to the present invention can be usedto generate polyclonal and/or monoclonal antibodies, anti-idiotypeantibodies thereto, or fragments thereof, which may used for diagnosticand/or therapeutic applications, according to known method steps, see,e.g., Harlow, supra, which is herein entirely incorporated by reference.

PP peptides or anti-idiotype antibodies (or fragments thereof) to PPpeptides are expected to be useful to quantitatively or qualitativelymodulate or prevent the development and/or symptoms associated withpsychoses and related disorders, such that administration of PP peptidesand/or anti-idiotype antibodies (or fragments thereof) may be used forresearch or therapeutic applications of the present invention.

Anti-PP antibodies (or fragments thereof) to PP peptides are alsoexpected to be useful to quantitatively or qualitatively modulate orprevent the development and/or symptoms associated with psychoses andrelated disorders, such that administration of anti-PP peptideantibodies (or fragments thereof) may be used for diagnostic or researchapplications of the present invention.

Such PP peptides, (including PP fragments, substitution derivatives andanti-idiotype antibody fragments) of the present invention may be usedto treat symptoms of, and provide treatment for, pathologies related topsychose and related disorders. D₂ receptor-related psychotic disorders,including schizophrenia, now treated with neuroleptics, are non-limitingexamples thereof.

The use of synthetic or recombinant PP peptides of the present inventioncan be preferable to the use of known drugs for schizophrenia andrelated disorders, e.g., which bind G-protein coupled receptors, such asneuroleptics that bind or inhibit the biological effect of binding toneuroreceptors as a non-limiting example. Such peptides are expected tohave significantly less side effects than presently used drugs presentlyused for treating schizophrenia and related disorders, includingneuroleptics, as they would structurally mimic naturally occurring PPpeptides and/or modulate abnormal ligand binding. Thus, PP peptides areexpected to have reduced side effects attributable to known foreigncompound drugs, with less immunogenicity, and reduced potential formotoric side effects (e.g., extrapyramidal symptoms and/or tardivedyskinesia).

The present invention is also related to the production, by chemicalsynthesis or recombinant DNA technology, of PP peptides, preferably assmall as possible while still retaining sufficient biological activityfor protecting or treating the effect on patients having symptomsrelated to schizophrenia or other psychoses.

PP peptides of the present invention may include fragments of 5-10 to50-150 amino acid fragments, or mutein sequences of PP peptides,including, e.g., homologs thereof having a homology of at least 80% withat least one PP peptide. See, e.g., Probst et al DNA and Cell Biology11:1-20 (1992), which is entirely incorporated herein by reference.

Also preferred are PP peptides corresponding to proteins whose encodingnucleic acid gene hybridizes to polynucleotide probes corresponding toSEQ ID NO:3, wherein the PP amino acid sequence is 10 to 1000 aminoacids in length, such as 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600,700, 800, 900 or 1000 amino acids, or any value or range therein.

An amino acid sequence of, or nucleic acid sequence encoding, a PPpeptide of the present invention is said to "substantially correspond"to another amino acid or nucleic acid sequence, respectively, if thesequence of amino acids or nucleic acid in both molecules provides PPpeptides having biological activity that is substantially similar inamino acid sequence of a PP peptide, such that only one to a few aminoacids differ in amino acid sequence. Additionally or alternatively, such"substantially corresponding" sequences of PP peptides includeconservative amino acid or nucleotide substitutions, or degeneratenucleotide codon substitutions wherein individual amino acid ornucleotide substitutions are well known in the art.

Accordingly, PP peptides of the present invention, or nucleic acidencoding therefor, include a finite set of substantially correspondingsequences as substitution peptides or polynucleotides which can beroutinely obtained by one of ordinary skill in the art, without undueexperimentation, based on the teachings and guidance presented herein.For a detailed description of protein chemistry and structure, seeSchulz, G. E. et al., Principles of Protein Structure, Springer-Verlag,New York, 1978, and Creighton, T. E., Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, 1983, which are herebyincorporated by reference. For a presentation of nucleotide sequencesubstitutions, such as codon preferences, see Ausubel et al, supra, at§§ A.1.1-A.1.24, and Sambrook et al, supra, at Appendices C and D.

Conservative substitutions of a PP peptide of the present inventionincludes a variant wherein at least one amino acid residue in the PPpeptide has been conservatively replaced by a different amino acid. Suchsubstitutions preferably are made in accordance with the following listas presented in Table II, which substitutions may be determined byroutine experimentation to provide modified structural and functionalproperties of a synthesized PP peptide molecule, while maintaining thepsychosis treating or protecting biological activity.

                  TABLE II    ______________________________________           Original     Exemplary           Residue      Substitution    ______________________________________           Ala          Gly;Ser           Arg          Lys           Asn          Gln;His           Asp          Glu           Cys          Ser           Gln          Asn           Glu          Asp           Gly          Ala;Pro           His          Asn;Gln           Ile          Leu;Val           Leu          Ile;Val           Lys          Arg;Gln;Glu           Met          Leu;Tyr;Ile           Phe          Met;Leu;Tyr           Ser          Thr           Thr          Ser           Trp          Tyr           Tyr          Trp;Phe           Val          Ile;Leu    ______________________________________

Alternatively, another group of substitutions of PP peptides of thepresent invention are those in which at least one amino acid residue inthe protein molecule has been removed and a different residue insertedin its place according to the following Table III. The types ofsubstitutions which may be made in the protein or peptide molecule ofthe present invention may be based on analysis of the frequencies ofamino acid changes between a homologous protein of different species,such as those presented in Table 1-2 of Schulz et al., supra, and FIGS.3-9 of Creighton, supra. Based on such an analysis, alternativeconservative substitutions are defined herein as exchanges within one ofthe following five groups:

TABLE III

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr(Pro, Gly);

2. Polar, negatively charged residues and their amides: Asp, Asn, Glu,Gln;

3. Polar, positively charged residues: His, Arg, Lys;

4. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys); and

5. Large aromatic residues: Phe, Tyr, Trp.

Conservative amino acid substitutions according to the presentinvention, e.g., as presented above, are known in the art and would beexpected to maintain biological and structural properties of a PPpeptide after amino acid substitution. Most deletions and insertions,and substitutions according to the present invention are those which donot produce radical changes in the characteristics of the protein orpeptide molecule. "Characteristics" is defined in a non-inclusive mannerto define both changes in secondary structure, e.g. α-helix or β-sheet,as well as changes in physiological activity, e.g. in receptor bindingassays.

However, when the exact effect of the substitution, deletion, orinsertion is to be confirmed one skilled in the art will appreciate thatthe effect of the substitution or substitutions will be evaluated byroutine screening assays, either immunoassays or bioassays to confirmbiological activity. For example, a substituted PP peptide typically ismade by site-specific mutagenesis of a PP peptide encoding nucleic acid,expression of the mutant nucleic acid in recombinant cell culture, and,optionally, purification from the cell culture, for example, byimmunoaffinity chromatography using a specific antibody on a chemicallyderivatized column or immobilized membranes or hollow fibers (to absorbthe mutant by binding to at least one epitope).

A preferred use of this invention is the production, by chemical orrecombinant DNA technology, of PP peptides, preferably as small aspossible while still retaining schizophrenia or related disordertreating or preventing biological activity.

Antibodies, Anti-Idiotype Antibodies and Fragments Thereof for PPPeptides of the Present Invention, and Proteins and Peptides RelatedThereto. This invention is also directed to antibodies ("Abs") orfragments thereof which bind at least one epitope specific for a PPpeptide of the present invention. The present invention is also directedto methods using such an antibody or fragment to detect the presence of,or measure the quantity or concentration of, a protein or polypeptidesharing at least one epitope with a PP peptide, the protein orpolypeptide being present in a cell, a cell or tissue extract, abiological fluid, an extract thereof, a solution, or sample, in vitro,in situ, or in vivo. Such methods provide a means to determine theextent, susceptibility or degree of psychosis or related disorders.

The term "anti-PP peptide antibody," or "anti-PP peptide Ab", is meantto encompass any antibody or fragment which specifically binds to any PPpeptide epitope, including polyclonal antibodies, monoclonal antibodies(mAbs), chimeric antibodies, anti-idiotypic (anti-Id) antibodies toantibodies specific for PP peptides of the present invention, as well asfragments, consensus polypeptides or chemical derivatives thereof (aspresented herein for PP peptides). Such anti-PP peptide Abs may beproduced by any known method steps, including hybridoma, recombinant orsynthetic production techniques. An antibody is said to be "capable ofbinding" a molecule if it is capable of specifically reacting with themolecule to thereby bind the molecule to the antibody. The term"epitope" is meant to refer to that portion of any molecule capable ofbeing bound by an antibody which can also be recognized by thatantibody. Epitopes or "antigenic determinants" usually consist ofchemically active surface groupings of molecules such as amino acids,lipids or sugar side chains and have specific three dimensionalstructural characteristics as well as specific charge characteristics.

An "antigen" is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce antibody capable of binding to an epitope of that antigen. Anantigen may have one, or more than one epitope. The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen. Amonoclonal antibody contains a substantially homogeneous population ofantibodies specific to antigens, which population contains substantiallysimilar epitope binding sites.

Anti-PP peptide antibodies may be obtained by any method steps known tothose skilled in the art. See, for example Kohler and Milstein, Nature256:495-497 (1975); U.S. Pat. No. 4,376,110; Ausubel et al, eds., supra;Sambrook, supra; Harlow, supra; and Coligan et al., supra, the contentsof which references are incorporated entirely herein by reference. Suchantibodies may be of any immunoglobulin class including IgG, IgM, IgE,IgA, GILD and any subclass thereof. A hybridoma producing a mAb of thepresent invention may be cultivated in vitro, in situ or in vivo.

Chimeric antibodies are molecules of which different portions arederived from different animal species, such as those having variableregion derived from a murine mAb and a human immunoglobulin constantregion, which are primarily used to reduce immunogenicity in applicationand to increase yields in production, for example, where murine mAbshave higher yields from hybridomas but higher immunogenicity in humans,such that human/murine chimeric mAbs are used. Chimeric antibodies andmethods for their production are known in the art (Cabilly et al, Proc.Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl.Acad. Sci. USA 81:6851-6855 (1984); Boulianne et al., Nature 312:643-646(1984); Cabilly et al., European Patent Application 125023 (publishedNov. 14, 1984); Neuberger et al., Nature 314:268-270 (1985); Taniguchiet al., European Patent Application 171496 (published Feb. 19, 1985);Morrison et al., European Patent Application 173494 (published Mar. 5,1986); Neuberger et al., PCT Application WO 86/01533, (published Mar.13, 1986); Kudo et al., European Patent Application 184187 (publishedJun. 11, 1986); Morrison et al., European Patent Application 173494(published Mar. 5, 1986); Sahagan et al., J. Immunol. 137:1066-1074(1986); Robinson et al., International Patent Publication No.PCT/US86/02269 (published May 7, 1987); Liu et al., Proc. Natl. Acad.Sci. USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad. Sci. USA84:214-218 (1987); Better et al., Science 240:1041-1043 (1988); andHarlow and Lane, supra. These references are incorporated entirelyherein by reference.

An anti-idiotypic (anti-Id) antibody is an antibody which recognizesunique determinants generally associated with the antigen-binding siteof an antibody. An Id antibody can be prepared by immunizing an animalof the same species and genetic type (e.g., mouse strain) as the sourceof the mAb with the mAb to which an anti-Id is being prepared. Theimmunized animal will recognize and respond to the idiotypicdeterminants of the immunizing antibody by producing an antibody tothese idiotypic determinants (the anti-Id antibody). See, for example,U.S. Pat. No. 4,699,880, which is herein entirely incorporated byreference. The anti-Id antibody may also be used as an "immunogen" toinduce an immune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may be epitopically identical tothe original mAb which induced the anti-Id. Thus, by using antibodies tothe idiotypic determinants of a mAb, it is possible to identify otherclones expressing antibodies of identical specificity. Accordingly, mAbsgenerated against a PP peptide of the present invention may be used toinduce anti-Id antibodies in suitable animals, such as BALB/c mice.Spleen cells from such immunized mice are used to produce anti-Idhybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs can becoupled to a immunogenic carrier such as keyhole limpet hemocyanin (KLH)or cationized bovine serum albumin and used to immunize additionalBALB/c mice. Sera from these mice will contain anti-anti-Id antibodiesthat have the binding properties of the original mAb specific for a PPpeptide epitope. The anti-Id mAbs thus have their own idiotypicepitopes, or "idiotopes" structurally similar to the epitope beingevaluated.

The term "antibody" is also meant to include both intact molecules aswell as fragments thereof, such as, for example, Fab and F(ab')₂, whichare capable of binding antigen. Fab and F(ab')₂ fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody(Wahl et al., J. Nucl. Med. 24:316-325 (1983)). It will be appreciatedthat Fab and F(ab')₂ and other fragments of the antibodies useful in thepresent invention may be used for the detection and quantitation of a PPpeptide according to the methods disclosed herein for intact antibodymolecules. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab')₂ fragments). See, e.g., Harlow, supra,Coligan, supra, Ausubel, supra. Additionally, synthetic or recombinantantibody fragments may be used which bind epitopes of PP peptides orrelated proteins.

Epitopes recognized by antibodies, and fragments and regions thereof, ofthe present invention may include 5 or more amino acids of a PP peptiderelated protein provided according to the present invention using probescorresponding to, or complementary to a 10-421 base sequence of SEQ IDNO:3, which a topographical epitope of a PP peptide or related proteinis recognized by, and specifically binds a anti-PP peptide antibody,fragments, and variable regions thereof.

The techniques to raise antibodies of the present invention to smallpeptide sequences that recognize and bind to those sequences in the freeor conjugated form or when presented as a native sequence in the contextof a large protein are well known in the art. Such antibodies includemurine, murine human and human-human antibodies produced by hybridoma orrecombinant techniques known in the art. See, Ausubel, supra, Harlow,supra, and Coligan, supra.

The identification of these peptide sequences recognized by mAbs of thepresent invention provides the information necessary to generateadditional monoclonal antibodies with binding characteristics andtherapeutic utility that parallel the embodiments of this application.

A PP-peptide specific murine, human or chimeric mAb of the presentinvention may be produced in large quantities by injecting hybridoma ortransfectoma cells secreting the antibody into the peritoneal cavity ofmice and, after appropriate time, harvesting the ascites fluid whichcontains a high titer of the mAb, and isolating the mAb therefrom. Forsuch in vivo production of the mAb with a non-murine hybridoma (e.g.,rat or human), hybridoma cells are preferably grown in irradiated orathymic nude mice.

Cell fusions for hybridoma formation of cells producing anti-PP peptideantibodies of the present invention may be accomplished by standardprocedures well known to those skilled in the field of immunology(Kohler and Milstein, Nature 256:495-497 (1975) and U.S. Pat. No.4,376,110; Hartlow, E. et al., supra; Campbell, A., "Monoclonal AntibodyTechnology," In: Laboratory Techniques in Biochemistry and MolecularBiology, Volume 13 (Burdon, R., et al., eds.), Elsevier, Amsterdam(1984); Kennett et al., Monoclonal Antibodies (Kennett et al., eds. pp.365-367, Plenum Press, N.Y., 1980); de St. Groth, S. F., et al., J.Immunol. Meth. 35: 1-21 (1980); Galfre, G. et al., Methods Enzymol.73:3-46 (1981); Goding, J. W. 1987. Monoclonal Antibodies: Principlesand Practice. 2nd ed. Academic Press, London, 1987);

Fusion partner cell lines and methods for fusing and selectinghybridomas and screening for mAbs are well known in the art (Hartlow, E.et al., supra; Kawamoto, T. et al., Meth. Enzymol 121:266-277 (1986);Kearney, J. F. et al., J. Immunol. 123:1548-1550 (1979); Kilmartin, J.V. et al., J. Cell Biol. 93:576-582 (1982); Kohler, G. et al., Eur. J.Immunol. 6:292-295 (1976); Lane, D. P. et al., J. Immunol. Meth.47:303-307 (1981); Mueller, U. W. et al., J. Immunol. Meth. 87:193-196(1986); Pontecorvo, G., Somatic Cell Genet. 1:397-400 (1975); Sharo, J.,et al., Proc. Natl. Acad. Sci. USA 76:1420-1424 (1979); Shulman, M. etal., Nature 276:269-270 (1978); Springer, T. A. (ed), HybridomaTechnology in the Biosciences and Medicine, Plenum Press, New York,1985; and Taggart, R. T. et al., Science 219:1228-1230 (1982)).

Alternatively, the antibodies my be produced by culturing hybridoma ortransfectoma cells in vitro and isolating secreted mAb from the cellculture medium.

PP peptide epitope related protein/gene detection and diagnosticmethods. Anti-PP peptide Abs and PP peptide encoding nucleic acid probesmay be used according to methods of the present invention to diagnosepatients having psychotic or related disorders, or to determine relativesubclinical and clinical degrees of such psychotic disorders, orpredisposition thereto. The present invention is based in part on thediscovery that PP peptide expression products, such as RNA and/or PPpeptides, have some protective effect on psychotic disorders in humansand possibly other mammals. Accordingly, the lack of, or presence of lowconcentrations of, PP peptide epitope containing peptides or proteins,or PP peptide encoding nucleic acids, such as mRNA, is expected tocorrelate with subclinical, clinical and/or acute psychoses and relateddisorders, such as schizophrenia.

Therefore, diagnostic and detection methods of the present inventionallow determination of the presence of, or susceptibility to, psychosesand related disorders in humans and mammals, using anti-PP peptide Absand/or PP peptide encoding nucleic acid probes.

Antibody detection of PP peptide epitope containing proteins. Antibodiesor fragments thereof having epitope binding sites specific for anepitope of a PP peptide, termed "anti-PP peptide antibodies," may beused to detect related or homologous proteins as a means to diagnose orprediagnose psychosis or related disorders, such as schizophrenia. Suchantibodies or fragments may thus be used to quantitatively orqualitatively detect a protein or peptide corresponding at least in partto a PP peptide in a sample or to detect the presence of such proteinsin biological fluids or cells which express such protein or peptide, invitro, in situ, or in vivo, based on the teaching and guidance presentedherein, without undue experimentation. The lack of, or presence of lowconcentrations of, PP peptide epitope containing peptides is expected tocorrelate with psychoses and related disorders, such as schizophrenia.

It will be appreciated that PP peptide antibodies, anti-idiotypeantibodies and fragments thereof, such as Fab and F(ab')₂, may be usedaccording to the present invention to detect and/or quantitate a PPpeptide according to the methods disclosed herein for intact antibodymolecules. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab')₂ fragments).

The antibodies of the present invention may be employed histologically,as in immunofluorescence or immunoelectron microscopy, for in situdetection of a PP peptide or a protein having psychosis protectingactivity. Through the use of such a procedure, it is possible todetermine not only the presence of a PP peptide or a protein havingpsychosis protecting activity, but also its distribution on the examinedtissue.

Additionally, the antibody of the present invention can be used todetect the presence of a soluble PP peptide or a protein havingpsychosis protecting activity, in a biological sample, such as a meansto monitor the presence and quantity of a PP peptide or a protein havingpsychosis protecting activity, used for diagnosis of the extent,susceptibility or degree of psychosis or related disorder.

Such immunoassays, for detecting a PP peptide, or a protein having a PPpeptide epitope, typically comprise incubating a biological sample, suchas a biological fluid, a tissue extract, freshly harvested cells such aslymphocytes or leukocytes, or cells which have been incubated in tissueculture, in the presence of a detectably labeled antibody capable ofidentifying a PP peptide, and detecting the antibody by any of a numberof techniques well-known in the art. See, e.g., Ausubel, supra, Harlow,supra.

The biological sample may be treated with a solid phase support orcarrier (which terms are used interchangeably herein) such asnitrocellulose, or other solid support which is capable of immobilizingcells, cell particles or soluble proteins. The support may then bewashed with suitable buffers followed by treatment with the detectablylabeled PP peptide-specific antibody. The solid phase support may thenbe washed with the buffer a second time to remove unbound antibody. Theamount of bound label on said solid support may then be detected byconventional means.

Such detection can be accomplished by any appropriate known method stepsfor detecting bound antibodies, such as enzyme linked immunosorbentassays (ELISA), isotope labeling, immunodiffusion assays, immunoaffinitychromatography, immunopreciptiation, protein staining, immunoblotting,iodination of proteins, biosynthetic labeling, or, e.g.,immunofluorescence techniques employing a fluorescently labeled antibody(see below) coupled with light microscopic, flow cytometric, orfluorometric detection. See, e.g., Coligan et al., supra, at Ch. 2, 5, 7and 8; Ausubel, supra, and Harlow, supra, which references are entirelyincorporated herein by reference.

By "solid phase support", "solid phase carrier", "solid support", "solidcarrier", "support" or "carrier" is intended any support or carriercapable of binding antigen or antibodies. Well-known supports orcarriers, include glass, polystyrene, polypropylene, polyethylene,dextran, nylon amylases, natural and modified celluloses,polyacrylamides, gabbros, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support or carrierconfiguration may be spherical, as in a bead, or cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, polymer teststrip, etc. Preferred supports or carriers include polystyrene beads.Those skilled in the art will know many other suitable carriers forbinding antibody or antigen, or will be able to ascertain the same byuse of routine experimentation. See Coligan, supra, at Ch. 8-9.

The binding activity of a given lot of anti-PP peptide antibody may bedetermined according to well known method steps. Those skilled in theart will be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation. See, e.g.,Harlow, supra, Coligan, supra, at Ch. 8. Other such steps as washing,stirring, shaking, filtering and the like may be added to the assays asis customary or necessary for the particular situation.

One of the ways in which a PP peptide-specific antibody, anti-idiotypeantibody or fragment thereof, can be detectably labeled is by linkingthe same to an enzyme and use in an enzyme immunoassay (EIA), accordingto known method steps. See Harlow, supra. Coligan, supra, at Ch.2.

Detection may be accomplished using any of a variety of otherimmunoassays. For example, by radioactivity labeling the antibodies orantibody fragments, it is possible to detect R-PTPase through the use ofa radioimmunoassay (RIA). A good description of RIA maybe found inLaboratory Techniques and Biochemistry in Molecular Biology, by Work etal., North Holland Publishing Company, N.Y. (1978) with particularreference to the chapter entitled "An Introduction to Radioimmune Assayand Related Techniques" by Chard, incorporated entirely by referenceherein. The radioactive isotope can be detected by such means as the useof a γ-counter, a scintillation counter or by autoradiography.

It is also possible to label an anti-PP peptide antibody, anti-idiotypeantibody or fragment thereof, with a fluorescent compound. When thefluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. Among themost commonly used fluorescent labelling compounds are fluoresceinisothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin,o-phthaldehyde and fluorescamine, commercially available, e.g., fromMolecular Probes, Inc. (Eugene, Oreg.). See, e.g., Ausubel, supra,Harlow, supra, Coligan, supra, at Ch. 2 and 5.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵² EU, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriamine pentaacetic acid (EDTA). See, e.g., Ausubel, supra,Harlow, supra, Coligan, supra, at § 5.3.

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

An antibody molecule of the present invention may be adapted forutilization in a immunometric assay, also known as a "two-site" or"sandwich" assay. In a typical immunometric assay, a quantity ofunlabeled antibody (or fragment of antibody) is bound to a solid supportor carrier and a quantity of detectably labeled soluble antibody isadded to permit detection and/or quantitation of the ternary complexformed between solid-phase antibody, antigen, and labeled antibody. See,e.g., Harlow, supra, and Coligan, supra, § 9.1.

Synthetic production of psychosis protecting peptides of the presentinvention. PP peptides and muteins can be synthesized according to knownmethod steps. Chemical polypeptide synthesis is a rapidly evolving areain the art, and methods of solid phase polypeptide synthesis arewell-described in the following references, hereby entirely incorporatedby reference: (Merrifield, B., J. Amer. Chem. Soc. 85:2149-2154 (1963);Merrifield, B., Science 232:341-347 (1986); Wade, J. D. et al.,Biopolymers 25:S21-S37 (1986); Fields, G. B., Int. J. Polypeptide Prot.Res. 35:161 (1990); MilliGen Report Nos. 2 and 2a, MilliporeCorporation, Bedford, Mass., 1987) Ausubel, supra, Sambrook et al,supra, Coligan, supra, Ch. 9, which references are all entirelyincorporated herein by reference.

In general, as is known in the art, such methods involve blocking orprotecting reactive functional groups, such as free amino, carboxyl andthio groups. After peptide bond formation, the protective groups areremoved (or de-protected). Thus, the addition of each amino acid residuerequires several reaction steps for protecting and deprotecting. Currentmethods utilize solid phase synthesis, wherein the C-terminal amino acidis covalently linked to an insoluble resin particle large enough to beseparated from the fluid phase by filtration. Thus, reactants areremoved by washing the resin particles with appropriate solvents usingan automated programmed machine. The completed polypeptide chain iscleaved from the resin by a reaction which does not affect polypeptidebonds.

More recently, the preferred "Fmoc" technique has been introduced as analternative synthetic approach, offering milder reaction conditions,simpler activation procedures and compatibility with continuous flowtechniques. This method was used, e.g., to prepare the peptide sequencesdisclosed in the present application. Here, the α-amino group isprotected by the base labile 9-fluorenylmethoxycarbonyl (Fmoc) group.The benzyl side chain protecting groups are replaced by the more acidlabile t-butyl derivatives. Repetitive acid treatments are replaced bydeprotection with mild base solutions, e.g., 20% piperidine indimethylformamide (DMF), and the final HF cleavage treatment iseliminated. A TFA solution is used instead to cleave side chainprotecting groups and the polypeptide resin linkage simultaneously.

At least three different polypeptide-resin linkage agents can be used:substituted benzyl alcohol derivatives that can be cleaved with 95% TFAto produce a polypeptide acid, methanolic ammonia to produce apolypeptide amide, or 1% TFA to produce a protected polypeptide whichcan then be used in fragment condensation procedures, as described byAtherton, E. et al., J. Chem. Soc. Perkin Trans. 1:538-546 (1981) andSheppard, R. C. et al., Int. J. Polypeptide Prot. Res. 20:451-454(1982). Furthermore, highly reactive Fmoc amino acids are available aspentafluorophenyl esters or dihydro-oxobenzotriazine esters derivatives,saving the step of activation used in the tBoc method.

Recombinant production of psychosis protecting peptides of the presentinvention. Sequences available to use as a basis for PP peptidesynthesis can be based on amino acid and/or nucleotide sequencescorresponding to SEQ ID NOs: 3-12. Recombinant production of PP peptidescan be accomplished according to known method steps. Standard referenceworks setting forth the general principles of recombinant DNA technologyinclude Watson, J. D. et al., Molecular Biology of the Gene, Volumes Iand II, The Benjamin/Cummings Publishing Company, Inc., publisher, MenloPark, Calif. (1987); Darnell, J. E. et al., Molecular Cell Biology,Scientific American Books, Inc., publisher, New York, N.Y. (1986);Lewin, B. M., Genes III, John Wiley & Sons, publishers, New York, N.Y.(1989); Old, R. W., et al., Principles of Gene Manipulation: AnIntroduction to Genetic Engineering, 2d edition, University ofCalifornia Press, publisher, Berkeley, Calif. (1981); Ausubel et al,eds., supra; Sambrook, supra; Harlow, supra; and Coligan et al., supra,the entire contents of which references are herein incorporated byreference.

A nucleic acid sequence encoding a PP peptide of the present inventionmay be recombined with vector DNA in accordance with conventionaltechniques, including blunt-ended or staggered-ended termini forligation, restriction enzyme digestion to provide appropriate termini,filling in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and ligation with appropriateligases. Techniques for such manipulations are disclosed, e.g., byAusubel et al, supra, and are well known in the art.

A nucleic acid molecule, such as DNA, is said to be "capable ofexpressing" a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are "operably linked" to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene expression as PP peptides inrecoverable amounts. The precise nature of the regulatory regions neededfor gene expression may vary from organism to organism, as is well knownin the analogous art. See, e.g., Sambrook, supra and Ausubel supra.

The present invention accordingly encompasses the expression of a PPpeptide, in either prokaryotic or eukaryotic cells, although eukaryoticexpression is preferred.

Preferred hosts are bacterial or eukaryotic hosts including bacteria,yeast, insects, fungi, bird and mammalian cells either in vivo, or insitu, or host cells of mammalian, insect, bird or yeast origin. It ispreferred that the mammalian cell or tissue is of human, primate,hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog or catorigin, but any other mammalian cell may be used.

Further, by use of, for example, the yeast ubiquitin hydrolase system,in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteinsmay be accomplished. The fusion proteins so produced may be processed invivo or purified and processed in vitro, allowing synthesis of a PPpeptide of the present invention with a specified amino terminussequence. Moreover, problems associated with retention of initiationcodon-derived methionine residues in direct yeast (or bacterial)expression may be avoided. Sabin et al., Bio/Technol. 7(7): 705-709(1989); Miller et al., Bio/Technol. 7(7): 698-704 (1989).

Any of a series of yeast gene expression systems incorporating promoterand termination elements from the actively expressed genes coding forglycolytic enzymes produced in large quantities when yeast are grown inmediums rich in glucose can be utilized to obtain PP peptides of thepresent invention. Known glycolytic genes can also provide veryefficient transcriptional control signals. For example, the promoter andterminator signals of the phosphoglycerate kinase gene can be utilized.

Production of PP peptides or functional derivatives thereof in insectscan be achieved, for example, by infecting the insect host with abaculovirus engineered to express at least one PP peptide by methodsknown to those of skill. See Ausubel, supra, at §§16.8-16.11.

In a preferred embodiment, the introduced nucleotide sequence will beincorporated into a plasmid or viral vector capable of autonomousreplication in the recipient host. Any of a wide variety of vectors maybe employed for this purpose. See, e.g., Ausubel et al, supra, §§ 1.5,1.10, 7.1, 7.3, 8.1, 9.6, 9.7, 13.4, 16.2, 16.6, and 16.8-16.11. Factorsof importance in selecting a particular plasmid or viral vector include:the ease with which recipient cells that contain the vector may berecognized and selected from those recipient cells which do not containthe vector; the number of copies of the vector which are desired in aparticular host; and whether it is desirable to be able to "shuttle" thevector between host cells of different species.

Preferred prokaryotic vectors known in the art include plasmids such asthose capable of replication in E. coli (such as, for example, pBR322,ColE1, pSC101, pACYC 184, πVX). Such plasmids are, for example,disclosed by Maniatis, T., et al. (Molecular Cloning, A LaboratoryManual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989); Ausubel et al, eds., supra. Bacillus plasmids includepC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T.(In: The Molecular Biology of the Bacilli, Academic Press, N.Y. (1982),pp. 307-329). Suitable Streptomyces plasmids include pIJ101 (Kendall, K.J., et al., J. Bacteriol. 169:4177-4183 (1987)), and streptomycesbacteriophages such as φC31 (Chater, K. F., et al., In: SixthInternational Symposium on Actinomycetales Biology, Akademiai Kaido,Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewedby John, J. F., et al. (Rev. Infect. Dis. 8:693-704 (1986)), and Izaki,K. (Jpn. J. Bacteriol. 33:729-742 (1978); and Ausubel et al, supra).

A gene or nucleic acid encoding for a naturally occurring protein havinga PP peptide sequence can also be detected, obtained and/or modified, invitro, in situ and/or in vivo, by the use of known DNA or RNAamplification techniques, such as PCR and chemical oligonucleotidesynthesis. PCR allows for the amplification (increase in number) ofspecific DNA sequences by repeated DNA polymerase reactions. Thisreaction can be used as a replacement for cloning, all that is requiredis a knowledge of the nucleic acid sequence. In order to carry out PCR,primers are designed which are complementary to the sequence ofinterest, such as a 10-140 base sequence as presented in SEQ ID NO:3.The primers are then generated by automated DNA synthesis. Becauseprimers can be designed to hybridize to any part of the gene, conditionscan be created such that mismatches in complementary base pairing can betolerated. Amplification of these mismatched regions can lead to thesynthesis of a mutagenized product resulting in the generation of apeptide with new properties (i.e., site directed mutagenesis). See also,e.g., Ausubel, supra, Ch. 16, and Coligan, supra, at §§ 10.20-10.23.Also, by coupling complementary DNA (cDNA) synthesis, using reversetranscriptase, with PCR, RNA can be used as the starting material forthe synthesis of the PP gene without cloning. Detection of PCR and othermethods of amplification of RNA and/or DNA are well known in the art andcan be used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.Known methods of DNA or RNA amplification include, but are not limitedto polymerase chain reaction (PCR) and related amplification processes(see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188,to Mullis et al.; 4,795,699 and 4,921,794 to Tabor et al; 5,142,033 toInnis; 5,122,464 to Wilson et al.; 5,091,310 to Innis; 5,066,584 toGyllensten et al; 4,889,818 to Gelfand et al; 4,994,370 to Silver et al;4,766,067 to Biswas; 4,656,134 to Ringold; and Innis et al eds. PCRProtocols: A Guide to Method and Applications) and RNA mediatedamplification which uses anti-sense RNA to the target sequence as atemplate for double stranded DNA synthesis (U.S. Pat. No. 5,130,238 toMalek et al, with the tradename NASBA); and immuno-PCR which combinesthe use of DNA amplification with antibody labeling (Ruzicka et al.,Science 260:487 (1993); Sano et al, Science 258:120 (1992); Sano et al.,Biotechniques 9:1378 (1991)), entire contents of which patents andreference are entirely incorporated herein by reference.

PP peptide antibody purification. The expressed protein may be isolatedand purified in accordance with known method steps, such as extraction,precipitation, chromatography, affinity chromatography, electrophoresis,or the like. For example, the cells may be collected by centrifugation,or with suitable buffers, lysed, and the protein isolated by columnchromatography, for example, on DEAE-cellulose, phosphocellulose,polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis orimmunoprecipitation. Alternatively, the PP peptide or mutein thereof maybe isolated by the use of anti-PP peptide antibodies. Such antibodiesmay be obtained by well-known methods, some of which are mentionedbelow. These antibodies may be immobilized on cellulose, agarose, hollowfibers, or cellulose filters by covalent chemical derivatives by methodswell known to those skilled in the art. See, e.g., Harlow, supra,Coligan, supra, Ausubel, supra.

As discussed herein, PP peptides of the present invention may be furthermodified for purposes of drug design, such as for example to reduceimmunogenicity, to prevent solubility and/or enhance delivery, or toprevent clearance or degradation.

Appropriate modification of the primary amino acid sequence of PPpeptides of the present invention, obtained by mutagenesis or utilizingfragments, as described herein, will allow the creation of moleculeswhich affect psychosis related symptoms than that exhibited by naturallypsychosis protecting proteins. Small polypeptides that are providedaccording to the present invention which polypeptides maintain psychosisprotecting activity, are expected to have two advantages over largerpolypeptides. These advantages include (1) greater stability anddiffusibility, and (2) less immunogenicity.

Pharmaceutical Preparations and Administration

Preparations of PP peptides for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions,which may contain auxiliary agents or excipients which are known in theart. Pharmaceutical compositions such as tablets and capsules can alsobe prepared according to routine methods.

By the term "protection" from infection or disease as used herein isintended "prevention," "suppression" or "treatment." "Prevention"involves administration of a PP peptide or anti-idiotypic antibody priorto the induction of the disease.

"Suppression" involves administration of the composition prior to theclinical appearance of the disease.

"Treatment" involves administration of the protective composition afterthe appearance of the disease. It will be understood that in human andveterinary medicine, it is not always possible to distinguish between"preventing" and "suppressing" since the ultimate inductive event orevents may be unknown, latent, or the patient is not ascertained untilwell after the occurrence of the event or events. Therefore, it iscommon to use the term "prophylaxis" as distinct from "treatment" toencompass both "preventing" and "suppressing" as defined herein. Theterm "protection," as used herein, is meant to include "prophylaxis."

At least one PP peptide, antibody or anti-idiotypic antibody of thepresent invention may be administered by any means that achieve theirintended purpose, for example, to treat PP related pathologies, such aspsychotic disorders, including schizophrenia using a PP peptide alone orpreferably in the form of a pharmaceutical composition.

For example, administration of such a composition may be by variousparenteral routes such as subcutaneous, intravenous, intradermal,intramuscular, intraperitoneal, intranasal, transdermal, or buccalroutes. Alternatively, or concurrently, administration may be by theoral route. Parenteral administration can be by bolus injection or bygradual perfusion over time.

A preferred mode of using a PP pharmaceutical composition of the presentinvention is by intravenous or parenteral application.

A typical regimen for preventing, suppressing, or treating schizophreniarelated symptoms or symptoms of other psychoses, comprisesadministration of an effective amount of a PP peptide administered overa period of one or several days, up to and including between one weekand about 24 months.

It is understood that the dosage of a PP peptide of the presentinvention administered in vivo or in vitro will be dependent upon theage, sex, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired. The ranges of effective doses provided below are not intendedto limit the inventors and represent preferred dose ranges. However, themost preferred dosage will be tailored to the individual subject, as isunderstood and determinable by one of skill in the art, without undueexperimentation.

The total dose required for each treatment may be administered bymultiple doses or in a single dose. A PP peptide or functional achemical derivative thereof may be administered alone or in conjunctionwith other therapeutics directed to schizophrenia related disorders orother symptoms of the disorder.

Effective amounts of the PP peptide or composition, or a PPanti-idiotypic antibody, are from about 0.01 μg to about 100 mg/kg bodyweight, and preferably from about 10 μg to about 50 mg/kg body weight,such 0.05, 0.07, 0.09, 0.1, 0.5, 0.7, 0.9, 1, 2, 5, 10, 20, 25, 30, 40,45, 50, 60, 70, 80, 90 or 100 mg/kg, or any value or range therein.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions, which may containauxiliary agents or excipients which are known in the art.Pharmaceutical compositions such as tablets and capsules can also beprepared according to routine methods.

Pharmaceutical compositions comprising at least one PP peptide of thepresent invention may include all compositions wherein the PP peptide iscontained in an amount effective to achieve its intended purpose. Inaddition to the PP peptide, a pharmaceutical composition may containsuitable pharmaceutically acceptable carriers, such as comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically.

Pharmaceutical compositions according to the present invention mayfurther, optionally comprise an antipsychotic, such as an therapeuticagent selected from the group consisting of a phenothiazine derivative,a thioxanthine derivative, a butyrophenone derivative, adihydroindolone, a dibenzoxazepine derivative and an atypicalneuroleptic (see, e.g., Baldessarini, supra, Katzung, supra).

Pharmaceutical compositions include suitable solutions foradministration intravenously, subcutaneously, dermally, orally,mucosally, rectally or may by injection or orally, and contain fromabout 0.01 to 99 percent, preferably from about 20 to 75 percent ofactive component (i.e. the antibody) together with the excipient.Pharmaceutical compositions for oral administration include tablets andcapsules. Compositions which can be administered rectally includesuppositories.

Transgenic Animals. Animal models of psychoses, such as schizophreniamay now be provided according to the present invention by the use oftransgenic animals that are inhibited (as psychosis model) orconsitutively express (as normal controls) PP peptide related proteins.

The present invention is thus also directed to a transgenic non-humaneukaryotic animal (preferably a rodent, such as a rat or mouse) whosegerm cells and somatic cells contain genomic DNA according to thepresent invention which codes for antisense or inhibiting expressionproducts which prevent the expression of PP peptide related proteinshaving a psychotic protecting effect in normal mammals. Such inhibitingnucleic acids may be introduced into the animal, or an ancestor of theanimal, at an embryonic stage, preferably the one-cell, or fertilizedoocyte, stage, and generally not later than about the 8-cell stage. Theactivated sequence, as the term is used herein, means a gene which, whenincorporated into the genome of the animal, is expressed in the animaland increases the probability of the development of a psychosis orrelated disorder in the animal.

There are several means by which such a inhibiting nucleic acid can beintroduced into the genome of the animal embryo so as to bechromosomally incorporated and expressed. One method is to transfect theembryo with the gene as it occurs naturally, and select transgenicanimals in which the gene has integrated into the chromosome at a locuswhich results in expression. Other methods for ensuring expressioninvolve modifying the gene or its control sequences prior tointroduction into the embryo. One such method is to transfect the embryowith a vector (see above) containing an already modified gene. Othermethods are to use a gene the transcription of which is under thecontrol of a inducible or constitutively acting promoter, whethersynthetic or of eukaryotic or viral origin, or to use a gene activatedby one or more base pair substitutions, deletions, or additions (seeabove).

Introduction of the desired gene sequence at the fertilized oocyte stageensures that the transgene is present in all of the germ cells andsomatic cells of the transgenic animal and has the potential to beexpressed in all such cells. The presence of the transgene in the germcells of the transgenic "founder" animal in turn means that all itsprogeny will carry the transgene in all of their germ cells and somaticcells. Introduction of the transgene at a later embryonic stage in afounder animal may result in limited presence of the transgene in somesomatic cell lineages of the founder; however, all the progeny of thisfounder animal that inherit the transgene conventionally, from thefounder's germ cells, will carry the transgene in all of their germcells and somatic cells.

Chimeric non-human mammals in which fewer than all of the somatic andgerm cells contain the desired PP peptide related protein inhibitingnucleic acid, produced, for example, when fewer than all of the cells ofthe morula are transfected in the process of producing the transgenicmammal, are also intended to be within the scope of the presentinvention.

The techniques described in Leder, U.S. Pat. No. 4,736,866, forproducing transgenic non-human mammals may be used for the production ofthe transgenic non-human mammal of the present invention. The varioustechniques described in Palmiter et al., Ann. Rev. Genet., 20, 465-99(1986), the entire contents of which are hereby incorporated byreference, may also be used.

The animals carrying this gene can be used to test compounds which mayaffect the progress of psychotic disorders or to test compounds whichmay be used to prevent the development of psychoses in susceptiblepatients. These tests can be extremely sensitive because of thepropensity of these transgenic animals to develop psychotic disorders.Such animals will also serve as an animal model enabling testing oftreatment and diagnostic methods for all psychotic disorders to beperformed on non-humans. Transgenic animals according to the presentinvention can also be used as a source of cells for cell culture.

Muteins of PP peptides of the present invention may include peptideswhich are distinct from PP peptides discussed above in criticalstructural features, but which maintain anti-schizophrenia biologicalactivity. Such consensus peptides may be derived by molecular modeling,optionally combined with hydrophobicity analysis and/or fitting to modelhelices, as non-limiting examples. Such modeling can be accomplishedaccording to known method steps using known modeling algorithms, suchas, but not limited to, ECEPP, INSIGHT, DISCOVER, CHEM-DRAW, AMBER,FRODO and CHEM-X.

Such consensus peptides or fragments of PPs may then be synthesized orproduced recombinantly, in order to provide PP peptides according to thepresent invention which have anti-schizophrenia or inhibit thebiological activity.

In addition, any amide linkage in any of the PP peptides can be replacedby a ketomethylene moiety, e.g. (--C(═O)--CH₂ --) for (--(C═O) --NH--).Such derivatives are expected to have the property of increasedstability to degradation by enzymes, and therefore possess advantagesfor the formulation of compounds which may have increased in vivo halflives, as administered by oral, intravenous, intramuscular,intraperitoneal, topical, rectal, intraocular, or other routes.

In addition, any amino acid representing a component of the saidpeptides can be replaced by the same amino acid but of the oppositechirality. Thus, any amino acid naturally occurring in theL-configuration (which may also be referred to as the R or S, dependingupon the structure of the chemical entity) may be replaced with an aminoacid of the same chemical structural type, but of the oppositechirality, generally referred to as the D- amino acid but which canadditionally be referred to as the R- or the S-, depending upon itscomposition and chemical configuration. Such derivatives have theproperty of greatly increased stability to degradation by enzymes, andtherefore are advantageous in the formulation of compounds which mayhave longer in vivo half lives, when administered by oral, intravenous,intramuscular, intraperitoneal, topical, rectal, intraocular, or otherroutes.

Additional amino acid modifications of amino acids of PP peptides of tothe present invention may include the following: Cysteinyl residues maybe reacted with alpha-haloacetates (and corresponding amines), such as2-chloroacetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteinyl residues may also bederivatized by reaction with compounds such as bromotrifluoroacetone,alpha-bromo- beta-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues may be derivatized by reaction with compounds such asdiethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain, and para-bromophenacyl bromide mayalso be used; e.g., where the reaction is preferably performed in 0.1 Msodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues may be reacted with compounds suchas succinic or other carboxylic acid anhydrides. Derivatization withthese agents is expected to have the effect of reversing the charge ofthe lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include compounds such asimidoesters/e.g., as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues may be modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin according to known method steps.Derivatization of arginine residues requires that the reaction beperformed in alkaline conditions because of the high pKa of theguanidine functional group. Furthermore, these reagents may react withthe groups of lysine as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues per se is well-known, suchas for introducing spectral labels into tyrosyl residues by reactionwith aromatic diazonium compounds or tetranitromethane. N-acetylimidizoland tetranitromethane may be used to form O-acetyl tyrosyl species and3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modifiedby reaction with carbodiimides (R'--N--C--N--R') such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4- dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues may be deamidated under mildly acidic conditions. Either formof these residues falls within the scope of the present invention.

Derivatization with bifunctional agents is useful for cross-linking thepeptide to a water-insoluble support matrix or to other macromolecularcarriers, according to known method steps. Commonly used cross-linkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such as methyl-3-(p-azidophenyl)dithio!propioimidate yield photoactivatable intermediatesthat are capable of forming crosslinks in the presence of light.Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 (which are herein incorporated entirely by reference), maybe employed for protein immobilization.

Other modifications of PP peptides of the present invention may includehydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl or threonyl residues, methylation of the alpha-amino groups oflysine, arginine, and histidine side chains (T. E. Creighton, Proteins:Structure and Molecule Properties, W.H. Freeman & Co., San Francisco,pp. 79-86 (1983)), acetylation of the N-terminal amine, methylation ofmain chain amide residues (or substitution with N-methyl amino acids)and, in some instances, amidation of the C-terminal carboxyl groups,according to known method steps.

Such derivatized moieties may improve the solubility, absorption,permeability across the blood brain barrier biological half life, andthe like. Such moieties or modifications of PP peptides mayalternatively eliminate or attenuate any possible undesirable sideeffect of the protein and the like. Moieties capable of mediating sucheffects are disclosed, for example, in Remington's PharmaceuticalSciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980).

Such chemical derivatives of PP peptides also may provide attachment tosolid supports, including but not limited to, agarose, cellulose, hollowfibers, or other polymeric carbohydrates such as agarose, cellulose,such as for purification, generation of antibodies or cloning; or toprovide altered physical properties, such as resistance to enzymaticdegradation or increased binding affinity or modulation for PP peptides,which is desired for therapeutic compositions comprising PP peptides,antibodies thereto or fragments thereof. Such peptide derivatives arewell-known in the art, as well as method steps for making suchderivatives using carbodiimides active esters of N-hydroxy succinimide,or mixed anhydrides, as non-limiting examples.

Variation upon consensus peptide sequences of PP peptide of the presentinvention may also include: the addition of one, two, three, four, orfive lysine, arginine or other basic residues added to the --COOHterminal end of the peptide; and/or one, two, three, four, or fiveglutamate or aspartate or other acidic residues added to the aminoterminal end of the peptide, where "acidic" and "basic" are as definedherein. Such modifications are well known to increase the a-helicalcontent of the peptide by the "helix dipole effect". They also canprovide enhanced aqueous solubility of the peptide. See, e.g., Baldwinet al., supra.

PP peptides of the present invention also include peptides havingun-natural amino acids by exploiting a phenomenon known as suppression.Some bacteria, when encountering a nonsense mutation (e.g., an internalstop codon: UAA, UAG, UGA) substitutes an amino acid using a chargedtransfer RNA that carries the proper anticodon to allow fortranslocation in spite of the error in RNA sequence (i.e. suppression).By charging the suppressor to RNA with an unnatural amino acid, peptidescan be generated with specific substitutions through modification duringtranslation.

Having now generally described the invention, the same will be morereadily understood through reference to the following example which isprovided by way of illustration, and is not intended to be limiting ofthe present invention.

EXAMPLE 1 Isolation of a PP Peptide Encoding Gene from Monozygotic TwinsDiscordant for Schizophrenia

Monozygotic twins discordant for schizophrenia represent excellentsubjects to assay for differences in gene expression at thetranscriptional level. The assumption is that variance in phenotype (inthis case mental function) is attributable to the way in which thegenetic potential is expressed. Removal of commonly expressedtranscripts by subtractive hybridization should result in enrichment ofphenotype specific gene products even when these transcripts are at lessthan 0.05% of the total in mRNA population (Travis et al, 1987).

The logical tissue source for these studies is the brain; however,because of our interest in living subjects, an alternative tissue, theperipheral blood lymphocyte was used. While seeming counter-intuitive,it is possible to show that gene expression in an accessible tissue inwhich a gene has no function may serve to monitor expression in aninaccessible tissue in which the function of the gene product hasphysiological significance (Chelly et al 1988). Accordingly, we havedemonstrated that a subtracted clone obtained from the lymphocytes of adiscordant schizophrenic twin pair is expressed in the CNS of rats.

Materials and Methods

The subjects for this study were 64 year old female monozygotic twinsdiscordant for schizophrenia (DSMIIIR). The schizophrenic co-twin hadbeen neuroleptic free for more than 30 years. Lymphocytes from 250 cc ofwhole blood were isolated by separation on Ficoll-Paque according to themanufacturers' instructions. RNA isolation, cDNA synthesis and cDNAcloning were accomplished as described by Belyavsky et al., Nucl. AcidsRes. 17:2919-2932 (1989), with minor modifications. A cDNA library wasmade for each twin and subtractive hybridization was achieved asdescribed in the manual provided in the Subtractor II Kit manufacturedby InVitrogen. Libraries were screened with (32p) labeled cDNA using the+/- method for differential clone identification and subsequentisolation. Probes for in situ hybridization were synthesized by in vitrotranscription with T7 (anti-sense) and SP6 RNA (sense) polymerases inthe presence of (33p) labeled UTP, using the subtracted clone as thetemplate. The use of the (33p) labeled results in greater resolution andshorter exposure time as compared to (35s) labeled isotopes.

RESULTS

A flow chart of the procedures used to detect the subtracted clones isshown in FIG. 2. Because we did not know, a priori, in which subject wewould observe differential expression, both libraries had to be used asdriver and substrate in two separate subtraction assays.

The number of subtracted clones identified in the assay where the "sick"twin's cDNA was used in excess was within the 2% background valuedetermined previously. However, when the cDNA of the "well" twin wasused in excess, the number of differential clones was approximately 4%.We isolated 41 clones for further analysis based on the results of theprimary screening. Secondary screening of the clones reduced the numberto 20. These twenty clones were then used for mini plasmid preparationsand subsequently sequenced. After sequencing the number of clones wasreduced to 10.

In order to verify that subtracted clones were differentially expressedwe employed an RNase protection assay (RPA). RPA results demonstratedthat out of the two clones tested thus far, one clone (pOKSC4c) wasdifferentially expressed. The expression of the clone was greatest forthe "well" twin.

The fact that this clone was expressed in the well twin but not the sickis consistent with our conclusion that his gene serves a protectivefunction in the well twin. If as shown earlier schizophrenia is agenetic disorder, both monozygotic twins carry the schizophrenia gene.The well twin expresses pOKSC4c while the schizophrenic twin does not;therefore pOKSC4c must be protecting the well twin from the deleteriouseffects of the schizophrenia gene.

In this case pOKSC4c is protecting against schizophrenia. In otherpatients genetically vulnerable to psychoses other than schizophrenia orvulnerable to other causes of psychosis, pOKSC4c could be expressed toprotect against these psychoses. Thus, although pOKSC4c was isolatedfrom twins discordant for schizophrenia, this in no way limits theprotective effect of PP peptide to schizophrenia.

In situ hybridization studies were carried out to determine if pOSKC4cwas expressed in rat brain. Examination of the in situ audioradiograph(FIG. 3A) reveals that the two most intense regions of hybridizationare: 1) the cortex and 2) the medial geniculate nucleus. CAI to CA3 ofAmmon's horn (hippocampus) also hybridize to the clone. Thehybridization signal in the cortex seems to be qualitatively higher incertain cortical regions. As seen in FIG. 3A, the interhinal cortex,perirhinal cortex and temporal cortex (areas 1 and 3) give a moreintense signal than the rest of the cortex. Lastly, these details werecompletely absent when the sense strand was used as a control (FIG. 3B).

DISCUSSION

Chelly et al., supra (1988), using quantitative PCR, found thatdystrophin message could be detected in lymphocytes from normal subjectsbut not in lymphocytes of subjects with Duchenne's muscular dystrophy.For the dystrophin gene, at least, a very low level of expression of thegene occurs in lymphocytes even though the gene product, dystrophin, hasa function in muscle but not in blood cells. Thus the dystrophin geneappears to "dribble" a very low level of RNA in the lymphocyte, whereasthe mutant gene does not "dribble" in lymphocytes of the DMD subjects.

As described above, the probe derived from lymphocytes via subtractivehybridization produces a strong signal in rat cortex and geniculatebody. The selective nature of the hybridization in rat brain supportsthe idea that this gene may be associated with specific functions in thebrain rather than be ubiquitously active.

Subtracted cDNA Clones from a Monozygotic Twin Pair Discordant forSchizophrenia pOKSC4c

pOKSC4c: The plasmid contains a 371 bp fragment, encoding what seems tobe the 3' end of a previously unreported gene, which has been insertedinto the BamH I site of the Invitrogen plasmid pcDNA II. The orientationof the insert is such that the SP6 promoter lies at the 5' end of thegene fragment.

This plasmid was isolated by screening a subtracted cDNA librarygenerated from the RNA of lymphocytes obtained from a set of monozygotictwins discordant for schizophrenia. The cDNA libraries used as substratefor the subtraction assay were constructed using PCR according to themethod described by Belyavsky, et al., Nucl. Acids Res. 17:2919-2932(1989), with slight modifications. The two oligonucleotides used inlibrary construction are described below.

Oligonucleotide used for first strand cDNA synthesis and 3 downstreamprimer during amplification for all cDNAs (5' to 3' orientation):TTTTTTTTTTTTTTTTCCCCGGGCTA* T7 promoter (SEQ ID NO:1)

SP6 promoter *ATCGAAATTCCCCCCCCCCCCC (SEQ ID NO:2)

Preliminary evidence using RNase protection assays provides the clearexpectation that the full length gene corresponding to the pOKSC4cinsert is differentially expressed in this set of twins, such that the"well twins" is expected to make significantly more of the proteinencoded by the full length gene than the "sick" co-twin.

Addititionally, data have been obtained which demonstrate that the genecorresponding to the pOKSC4c fragment is expressed in rat brain.Characterization of the full length gene accordingly may be providedbased on the use of probes based on or derived from SEQ ID NO:3according to known method steps, without undue experimentation (see,e.g., Sambrook, supra, Ausubel, supra). It is also expected thatfragments of this gene corresponding to the protein coding region may beused according to the present invention to provide a marker for variouspsychoses, such as schizophrenia, as well as a means to treat suchdisorders.

EXAMPLE II Cloning and Expression of PP Peptide Related Proteins

According to the present invention, 15-45 base portions of SEQ ID NO:3are used as oligo probes to screen genomic and cDNA libraries accordingto known method steps as presented in Ausubel, supra, and Sambrook,supra. Isolated clones are then expressed in suitable expression vectorsin appropriate host cells and sequenced as both the DNA encoding thegene and the expressed PP peptide related protein. The protein is thenpurified and sequenced, and then used to generate antibodies, togenerate transgenics expressing and not expressing the PP peptide orrelated protein, as animal models of psychoses, and as part oftherapeutic compositions used for treating various psychotic disorders,such as schizophrenia, as described herein.

EXAMPLE III Subtracted Clones Containing DNA Encoding PP Peptide RelatedProtein

According to the methods presented in Examples I and II above, andaccording to method steps known in the art, the following clones wereisolated and sequenced: POKSC4c (371 bp)

This cDNA seems to be expressed in the "well" twin more abundantly thanin the "sick" co-twin as determined by RPA.

This cDNA has also been shown to be expressed in the following areas ofthe rodent brain; cortex (RPA, Northern blot and In situ Hybridization),hippocampus (RPA and In situ hybridization) and medial geniculatenucleus (In situ hybridization).

TTTTCAGCAG TTGGCCTTTG TTGAGAAAAT GTGTGACTTT GCCCAAGCCC AGTAACTTGG 60AGCCTTGAAT TTGAGATGCT GGAAAGGGAG TCCTTCCTCC TTTCTGCAGT GTTGTCCCTA 120GTTTACCAAA GTCCATTTTG AATGTACCAT CCCCAGCCCA ACTCCAGCCT ACAGATAGTG 180CCAGACCGCC AGTAGGTGAG TAGCACTGTC TTCCTGGTCC GGACCTAGTG GCTTTGCTGT 240TAGGACCTTA TCACTAGAGA TGGCCTGGAT TTAGAGACGA ACCATTAGCT GTTGCCATAG 300CTGTTGCCAT ATGATTACGT GGCCTTGGGT TTCGCAATAA TTTATTTGGG TTCACTAAAT 360TTTAAATTTC T 371 (SEQ ID NO:3)

POKSC25b (206 bp)

This cDNA seems to be expressed in the "well" twin more abundantly thanin the "sick" cotein as determined by RPA.

TTCGTATGGA CTCGGAATAA AGTAAGTGTT TTCAGCCTGG CTGGATGTAT GTTGCAAAAT 60GGCCTCGATT CACCCAGGGC AATAAACAGT GGTATTGATA ACCCAAAACA ATAGTAATTG 120AAAATAATTT GTTTTAAAAG TATATGCTTT TCTTTTGATA CTCAAGTGTT TCATATTAGA 180GGTAAATGAG AAAATATAGA TGAACC 206 (SEQ ID NO:4)

pOKSC27c (110 bp)

TTTGGAAGAT TTATTAATTG ATTAAGGACT AGGAGGTCCA GCTAAAATGC AATTGGATTT 60ATTAAGGTAC TTAAATCCAG ATTTAAGGTA TGAAATCAAG AATGGCGAAC 110 (SEQ ID NO:5)

Seems to be expressed in the "well" twin more abundantly than in the"sick" co-twin as determined by RPA. Preliminary analysis suggests thatit is also expressed in rat brain (Northern blot).

TCCAAGAACA GTTTTGGGCC AAACAGACGA ACAGCCAGTT GGTTTTCTAT ACCAACTGTG 60TGATTTATTA GAGCTGTCAC ATGCTATCAT GAACTGCGCT GTACGAAGTC AACTCAGTGA 120TGATGTGAGG TATCACTACT AGTTGGTTGG TTGGTTACAT ATCTTAAATA TGTAGACAAC 180TTACCAACTG GAATGTTCAG CTAGCTAATA TCTCAATTAG AATCCATCTC ACTAGGAATG 240GGCAAACACT TGTGTTCTAA AGTTACTTGA AAGTAGTTTA TACTGCCAAC TTGATATATA 300TCATGCTATA GTTTGAACAT TTTGTGTACT TCCAAAATTC ACATTAAAAT CTAATCCAC 359(SEQ ID NO:6)

TTTAGTTAGC TGGGCCTACA GGCATGCACC ACCACCATTG GCTAAGTTTT GTATTTTCAG 60TAGAGACAGG GTTCACCATG TTGGCCAGGC TGCTCTTGAA CTCCTGACCT CAAGTGATCC 120ACCTACCTCC GCCTCCGAAA AGTGCTAGGA TTACAGGCCT GAGCCACTGT GCCTGGCCTG 180ATAAAGCACA TTTAAAGATC T 201 (SEQ ID NO:7)

pOKSC18a (45 bp)

ATTGCACTCC ATCCAGCCTG GGCAACAAGA GAGAAACTCC ATCTC 45 (SEQ ID NO:8)

pOKSC37a (174 bp)

TGTCTCTAGT AAAAATACAA AAATTGGCCG AGCGTGAAGG CTGGCGCCTC TAATCCCAGC 60TTCTTGGGAA GCTGAGGGAA GCTGAGGCAC AAGAATTTGC TTGAGCCCAC GAGTGGTTGA 120ATGCCAGGAC CTGTCCACTG CACTCCAGCC TGGGCGACAG AACGACACTG TCTC 174 (SEQ IDNO:9)

pOKSC41a (212 bp)

GTCTGGAGTT CAAAACCATC CTGGCATTTA TGGTGAAACC CTGTCTCTAC TAAAAATACA 60AAATAGACAG GTGTGGGTGT CACGCCTGTA GTCCCAGCTA CTCGGAAGGC TGAGGCAGGA 120GAATCGCTTG AACCTGGGAG GCAGAGGTTG CATTGAGGCA AGATCGCACC ACTGTACTCC 180AGCCAGGGTG ACAGAGCGGG ACTCTGTCAT TT 212 (SEQ ID NO:10)

pOKSC6f (247 bp)

This clone has identity with human beta globin region on chromosome 11,as having a 61.3% identity in 173 bp overlap.

ATGTTATCCC TTGAATGTAG TGTGTAACAG AGAGAGATGT TTCTTTCTTT CTTTGATTAT 60CTGAGAAGCT AGGCAGGTGA AAGAACTTTC TTGTCCTCCA TTCAGAAATA ATTTACAGGC 120AGTTACTTCT AAATATGCAT GCCTGGGCCA AATGTGGTGG CTCACACCTG TAATCCCAAC 180CCTGGGAAGC TGAGGCAGGA GGATTGCTTG CAACCAGCCT GGGTAGACAT AGTGAAACCT 240GTCTCTC 247 (SEQ ID NO:11)

pOKSC8a (32 bp)

AGCCTGGGCG ACAGAGAGCC AAACGCCGTC TG 32 (SEQ ID NO:12)

In the above sequences and description, bp=base pairs, RPA=RNaseProtection Assay and PCR=Polymerase Chain Reaction.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 12    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 26 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ    #              26  CCCC GGGCTA    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ    #                 22CCC CC    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 371 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:3: (xi) SEQUENCE DESCRIPTION: SEQ    - TTTTCAGCAG TTGGCCTTTG TTGAGAAAAT GTGTGACTTT GCCCAAGCCC AG - #TAACTTGG      60    - AGCCTTGAAT TTGAGATGCT GGAAAGGGAG TCCTTCCTCC TTTCTGCAGT GT - #TGTCCCTA     120    - GTTTACCAAA GTCCATTTTG AATGTACCAT CCCCAGCCCA ACTCCAGCCT AC - #AGATAGTG     180    - CCAGACCGCC AGTAGGTGAG TAGCACTGTC TTCCTGGTCC GGACCTAGTG GC - #TTTGCTGT     240    - TAGGACCTTA TCACTAGAGA TGGCCTGGAT TTAGAGACGA ACCATTAGCT GT - #TGCCATAG     300    - CTGTTGCCAT ATGATTACGT GGCCTTGGGT TTCGCAATAA TTTATTTGGG TT - #CACTAAAT     360    #      371    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 206 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:4: (xi) SEQUENCE DESCRIPTION: SEQ    - TTCGTATGGA CTCGGAATAA AGTAAGTGTT TTCAGCCTGG CTGGATGTAT GT - #TGCAAAAT      60    - GGCCTCGATT CACCCAGGGC AATAAACAGT GGTATTGATA ACCCAAAACA AT - #AGTAATTG     120    - AAAATAATTT GTTTTAAAAG TATATGCTTT TCTTTTGATA CTCAAGTGTT TC - #ATATTAGA     180    #             206  TAGA TGAACC    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 110 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:5: (xi) SEQUENCE DESCRIPTION: SEQ    - TTTGGAAGAT TTATTAATTG ATTAAGGACT AGGAGGTCCA GCTAAAATGC AA - #TTGGATTT      60    #             110ATCCAG ATTTAAGGTA TGAAATCAAG AATGGCGAAC    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 359 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:6: (xi) SEQUENCE DESCRIPTION: SEQ    - TCCAAGAACA GTTTTGGGCC AAACAGACGA ACAGCCAGTT GGTTTTCTAT AC - #CAACTGTG      60    - TGATTTATTA GAGCTGTCAC ATGCTATCAT GAACTGCGCT GTACGAAGTC AA - #CTCAGTGA     120    - TGATGTGAGG TATCACTACT AGTTGGTTGG TTGGTTACAT ATCTTAAATA TG - #TAGACAAC     180    - TTACCAACTG GAATGTTCAG CTAGCTAATA TCTCAATTAG AATCCATCTC AC - #TAGGAATG     240    - GGCAAACACT TGTGTTCTAA AGTTACTTGA AAGTAGTTTA TACTGCCAAC TT - #GATATATA     300    - TCATGCTATA GTTTGAACAT TTTGTGTACT TCCAAAATTC ACATTAAAAT CT - #AATCCAC     359    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 201 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:7: (xi) SEQUENCE DESCRIPTION: SEQ    - TTTAGTTAGC TGGGCCTACA GGCATGCACC ACCACCATTG GCTAAGTTTT GT - #ATTTTCAG      60    - TAGAGACAGG GTTCACCATG TTGGCCAGGC TGCTCTTGAA CTCCTGACCT CA - #AGTGATCC     120    - ACCTACCTCC GCCTCCGAAA AGTGCTAGGA TTACAGGCCT GAGCCACTGT GC - #CTGGCCTG     180    #                 201TC T    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 45 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:8: (xi) SEQUENCE DESCRIPTION: SEQ    #45                CCTG GGCAACAAGA GAGAAACTCC ATCTC    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 174 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:9: (xi) SEQUENCE DESCRIPTION: SEQ    - TGTCTCTAGT AAAAATACAA AAATTGGCCG AGCGTGAAGG CTGGCGCCTC TA - #ATCCCAGC      60    - TTCTTGGGAA GCTGAGGGAA GCTGAGGCAC AAGAATTTGC TTGAGCCCAC GA - #GTGGTTGA     120    - ATGCCAGGAC CTGTCCACTG CACTCCAGCC TGGGCGACAG AACGACACTG TC - #TC     174    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 212 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:10:(xi) SEQUENCE DESCRIPTION: SEQ    - GTCTGGAGTT CAAAACCATC CTGGCATTTA TGGTGAAACC CTGTCTCTAC TA - #AAAATACA      60    - AAATAGACAG GTGTGGGTGT CACGCCTGTA GTCCCAGCTA CTCGGAAGGC TG - #AGGCAGGA     120    - GAATCGCTTG AACCTGGGAG GCAGAGGTTG CATTGAGGCA AGATCGCACC AC - #TGTACTCC     180    #         212      CGGG ACTCTGTCAT TT    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 247 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:11:(xi) SEQUENCE DESCRIPTION: SEQ    - ATGTTATCCC TTGAATGTAG TGTGTAACAG AGAGAGATGT TTCTTTCTTT CT - #TTGATTAT      60    - CTGAGAAGCT AGGCAGGTGA AAGAACTTTC TTGTCCTCCA TTCAGAAATA AT - #TTACAGGC     120    - AGTTACTTCT AAATATGCAT GCCTGGGCCA AATGTGGTGG CTCACACCTG TA - #ATCCCAAC     180    - CCTGGGAAGC TGAGGCAGGA GGATTGCTTG CAACCAGCCT GGGTAGACAT AG - #TGAAACCT     240    #         247    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 32 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    #ID NO:12:(xi) SEQUENCE DESCRIPTION: SEQ    #          32      AGCC AAACGCCGTC TG    __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid molecule comprising atleast 30 consecutive nucleotides from SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, or SEQ ID NO:9, or from the complementary strand of any onethereof.
 2. A nucleic acid molecule in accordance with claim 1,comprising 30-100 consecutive nucleotides from SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, or SEQ ID NO:9, or from the complementary strand of any onethereof.
 3. An isolated nucleic acid molecule comprising at least 30consecutive nucleotides from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, orSEQ ID NO:9, or from the complementary strand of any one thereof, andfurther comprising a detectable label such that said molecule can beused as a detectable probe.
 4. An isolated molecule comprising a humancDNA molecule which includes the sequence of SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:9, or the complementary strand ofany one thereof.
 5. An isolated protein encoded by a human cDNA moleculein accordance with claim
 4. 6. A vector comprising a nucleic acidmolecule comprising at least 30 consecutive nucleotides from SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:9, or from thecomplementary strand of any one thereof.
 7. A vector in accordance withclaim 6, wherein said nucleic acid molecule comprises 30-100 consecutivenucleotides from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:9,or from the complementary strand of any one thereof.
 8. A vectorcomprising a nucleic acid molecule in accordance with claim
 4. 9. A hostcell comprising a vector in accordance with claim
 6. 10. A host cellcomprising a vector in accordance with claim
 7. 11. A host cellcomprising a vector in accordance with claim
 8. 12. A nucleic acidmolecule in accordance with claim 1, comprising at least 30 consecutivenucleotides from SEQ ID NO:3, or from the complementary strand thereof.13. A nucleic acid molecule in accordance with claim 12 comprising30-100 consecutive nucleotides from SEQ ID NO:3, or from thecomplementary strand thereof.
 14. A nucleic acid molecule in accordancewith claim 12, further comprising a detectable label such that saidmolecule can be used as a detectable probe.
 15. A molecule in accordancewith claim 4 comprising a human cDNA molecule which includes thesequence of SEQ ID NO:3, or the complementary strand thereof.
 16. Anisolated protein encoded by a human cDNA molecule in accordance withclaim
 15. 17. A vector comprising a nucleic acid molecule in accordancewith claim
 12. 18. A vector comprising a nucleic acid molecule inaccordance with claim
 13. 19. A vector comprising a nucleic acidmolecule in accordance with claim
 15. 20. A host cell comprising avector in accordance with claim
 17. 21. A host cell comprising a vectorin accordance with claim
 18. 22. A host cell comprising a vector inaccordance with claim
 19. 23. An isolated nucleic acid moleculecomprising at least 60 consecutive nucleotides from SEQ ID NO:6, or fromthe complementary strand thereof, and further comprising a detectablelabel such that said molecule can be used as a detectable probe.
 24. Avector comprising a nucleic acid molecule comprising at least 60consecutive nucleotides from SEQ ID NO:6, or from the complementarystrand thereof.
 25. A vector comprising a nucleic acid moleculecomprising 60-100 consecutive nucleotides from SEQ ID NO:6, or from thecomplementary strand thereof.
 26. A host cell comprising the vector inaccordance with claim
 24. 27. A host cell comprising the vector inaccordance with claim 25.